1. Field
This invention relates generally to the field of radioimmunoassay and specifically to solid-phase radioimmunoassay.
Radioimmunoassay (RIA) is a term used to describe any of several methods for determining very small concentrations of substances (especially in biological fluids), which methods are based on the use of radioactively labelled substances which can form immunochemical complexes with antibodies to that substance. Various RIA techniques are known for measuring concentrations of both antibodies and substances for which there exist antibodies. The RIA of a substance for which there exists antibodies (specific to that substance) is based on the observation that a known amount of that substance which has been radioactively labelled will tend to compete equally with an unknown amount of that substance (unlabelled) for a limited number of complexing sites on antibodies specific to the substance. Thus, a RIA of a given substance is performed as follows: a known amount of labelled substance and the unknown amount of that substance (unlabelled) are incubated with antibodies specific to the substance. During incubation, immunochemical complexes of both antibody-substance (labelled) and antibody-substance (unlabelled) are formed. The complexes are then removed from the incubation solution. Radioactivity counts are then taken of either the removed complexes or the remaining solution. The counts can be used to determine the unknown concentration by relating the counts to standard curves prepared beforehand using constant known amounts of labelled substance and known amounts of unlabelled substance.
An essential step in RIA methods in the separation of complexed products from the incubation solution. Ideally, the separation should be relatively simple and quick. It has been recognized that separation is greatly facilitated by using antibodies which have been immobilized by attachment to essentially water-insoluble carrier materials. When such carrier materials are used in RIA, the technique is commonly referred to as solid phase RIA, or SPRIA.
2. Prior Art:
Antibodies, antigenic substances, and other substances such as enzymes have been attached by covalent bonds to various organic substances as shown by U.S. Pat. No. 3,555,143 (proteins), issued to Axen et al. on Jan. 12, 1971, U.S. Pat. No. 3,645,852 (proteins), issued to Axen et al. on February 29, 1972, and U.S. Pat. No. 3,278,392 (enzymes), issued to Patchornik on Oct. 11, 1966. Although the above disclosures indicate the utility of the attached substances is not substantially impaired in the immobilization process, it has been found that, in many instances organic carrier materials have certain inherent disadvantages. For example, many organic carrier materials are subject to microbial attack, difficult to sterilize, difficult to comminute, and may not permit proper diffusion of reactants in certain environments. These and other disadvantages hve been overcome to a great extent by recent developments showing that inorganic materials can be used as carriers for the immobilized substances.
Antibodies, antigenic substances, and enzymes have been attached by covalent bonds to various inorganic materials through intermediate silane coupling agents as shown by U.S. Pat. No. 3,652,761 (antibodies and antigens), issued to H. H. Weetall on Mar. 28, 1972, U.S. Pat. No. 3,519,538 (enzymes), issued to R. A. Messing and H. H. Weetall on July 7, 1970, and U.S. Pat. No. 3,669,841 (enzymes),issued to R. E. Miller on June 11, 1972. In U.S. Pat. application Ser. No. 377,625, filed July 9, 1973 in the names of H. H. Weetall and S. Yaverbaum, and assigned to the present assignee, there is disclosed a method for extracting insulin from a solution which involves using a composite consisting of anti-insulin antibodies coupled to porous glass particles which, prior to coupling, had been surface activated with a cyanogen bromide solution. Various inorganics have also been used as sorbants in the RIA of substances such as thyroid hormones as shown by U.S. Pat. No. 3,666,854 (separating complexed hormone from free hormone by adsorbing the free hormone on various colloidal inorganics), issued on May 30, 1972 to A. M. Eisentraut, and U.S. Pat. No. 3,776,698 (using various inorganics to adsorb thyroid hormones after separation from a hormone-antibody complex), issued Dec. 4, 1973 to A. M. Eisentraut. A very recent article by W. F. Line et al. in Clin. Chem., 19, No. 12, pp. 1361-65 (1973) describes a SPRIA for digoxin substantially identical to one embodiment of our invention. See also an article by F. H. Bodley et al. in Steroids, Vol. 21, No. 1, pp. 1-16 (1973) wherein a similar SPRIA for progesterone is disclosed.
Although the above-cited U.S. Pat. No. 3,652,761 shows that various antibodies can be successfully coupled through intermediate silane coupling agents to a wide variety of inorganic materials, and that the resulting composites can be used to isolate the corresponding antigen from a solution, that patent broadly discloses the isolation of antigens using such composites. We have found that by making certain critical modifications of the broad teachings of U.S. Pat. No. 3,652,761, it is possible to prepare immunochemical composites which can be successfully used to provide an accurate and quick SPIRA for a number of substances in clinically significant concentration ranges. Our composites and the methods of using them in SPIRA are described in detail herein.